FOR RESEARCH USE ONLY!
Gelatinase (Gelatin Degradation/Zymography) Assay Kit (Fluorometric) 03/17
(Catalog # K444-100; 100 assays, Store kit at -20°C)
I.
Introduction:
Gelatinases are a type of matrix zinc-dependent metalloproteases (MMPs) that degrade gelatins and a variety of other extracellular matrix
proteins. These enzymes are synthesized as latent zymogens that are activated by proteolysis and inhibited by tissue inhibitors of
metalloproteases (TIMPs). Two mammalian gelatinases, Gelatinase A (MMP-2) and Gelatinase B (MMP-9), are critical for basement
membrane degradation and are highly upregulated in variety of tumor cells. Gelatinase activity is usually detected by small peptide-based
activity assays which may suffer from lack of substrate specificity. Other methods for gelatinase activity include gelatin Zymography where
samples are electrophoresed on a gelatin-containing SDS-PAGE, and further renatured in a suitable buffer for 12-16 h. The zymogram is
subsequently stained, and areas of digestion appear as clear bands against a darkly stained background where the substrate has been
degraded by the enzyme. Such methods are laborious, time-consuming and may lead to the loss of enzymatic activity as renaturation may
not be completely reversible. BioVision’s Gelatinase Activity Assay Kit utilizes a hybrid approach for the detection of gelatinase activity by
employing a highly quenched gelatin substrate which upon cleavage by a suitable gelatinase releases a fluorophore, which can be easily
quantified using a conventional microplate reader. This method is substrate-specific, simple, fast, high-throughput adaptable and amenable
to the sensitive detection of gelatinase activity (as low as 0.06 mCDU for bacterial collagenase) in biological samples.
Gelatinase Substrate
Gelatinase
Cleaved Gelatin + FITC Fluorescence (Ex/Em = 490/520 nm)
II.
Applications:
• Detection of gelatinase activity in biological samples such as tissue, cell lysates, etc.
III.
Sample Type:
• Recombinant protein, tissue, cell lysates, etc.
IV.
Kit Contents:
Components
Gelatinase Assay Buffer
Cell Lysis Buffer
Enzyme Positive Control
Gelatinase Substrate
FITC Standard (5 mM)
K444-100
Cap Code
Part Number
25 ml
25 ml
10 µl
1 Vial
10 µl
WM
NM
Green
Red
Yellow
K444-100-1
K444-100-2
K444-100-3
K444-100-4
K444-100-5
V.
User Supplied Reagents and Equipment:
• 96-well Clear/Black/White well plate (The black plate will yield lowest background while white plate will yield highest background
fluorescence).
• Multi-well spectrofluorometer
VI.
Storage Conditions and Reagent Preparation:
Store the entire kit at -20°C, protected from light. Briefly centrifuge small vials at low speed prior to opening. Read the entire protocol before
performing the experiment.
• Gelatinase Assay Buffer: Bring to room temperature before use. Store at -20°C.
• Gelatinase Substrate: Reconstitute in 220 µl of DI water. Mix well by pipetting up and down. Vortex if necessary. Unused substrate can
be stored at -20°C by covering it with aluminum foil or transferring it to an amber vial.
• Enzyme Positive Control: Aliquot and store at -20°C. Thaw on ice before use. Avoid repeated freeze/thaw.
VII.
Gelatinase Assay Protocol:
1. Sample Preparation: Homogenize fresh or frozen tissue (~5-10 mg) or cells (1-2 x 106) with 100 µl Cell Lysis Buffer and incubate on ice
for 5 min. Centrifuge the homogenate at 16,000 X g, 4°C for 10 min. Transfer the clarified supernatant to a fresh pre-chilled tube and
keep on ice. Measure the amount of protein in the lysate or purified enzyme using BCA Protein Assay Kit - Reducing Agent Compatible
(Cat. K818-1000 or equivalent). Add 1-50 μl of lysate or purified enzyme into desired well(s) in a white 96-well plate. If necessary, dilute
the lysate with Gelatinase Assay buffer. For Positive Control, dilute 2 µl of Enzyme Positive Control with 18 µl of Gelatinase Assay Buffer
and use 1-10 μl/well. Adjust the volume of Samples and Positive Control to 50 μl/well with Gelatinase Assay Buffer.
Notes:
a. The kit is designed to work with active Gelatinase enzymes only. If the sample contains inactive zymogen forms of gelatinase, it can
be activated with p-aminophenylmercuric acetate (APMA) or other activators. The conditions for activation of each enzyme should be
determined empirically by following appropriate testing protocol (Shapiro et. al., J. Bio. Chem. 1995, 270 (11), 6351-6356).
b. We recommend using the tissue/cell homogenate immediately to measure the Gelatinase activity. If desired, snap freeze the lysate
and store at -80°C.
c. For unknown samples, we suggest doing pilot experiment and testing 3-5 different amounts of samples to ensure the readings are
within the Standard Curve range.
d. To induce higher gelatinase expression, cells can also be grown in the presence of Phorbol myristate acetate (10-50 ng/ml), lysed and
tested directly in the assay (Shin et. al., Exp. Mol. Med., 2003, 39 (1), 97-105).
155 S. Milpitas Blvd., Milpitas, CA 95035 USA | T: (408)493-1800 F: (408)493-1801 | www.biovision.com | [email protected]
FOR RESEARCH USE ONLY!
e. Optional: For samples having background, prepare parallel sample well(s) as sample background control. Use same amount of
tissue/cell homogenate or purified enzyme as in the sample well. Adjust the final volume to 100 µl with Gelatinase Assay Buffer.
2. Standard Curve Preparation: Prepare 50 µM of FITC Standard by diluting 2 µl of 5 mM FITC Standard to 200 µl of Gelatinase Assay
Buffer. Mix well by pipetting up and down, vortex vigorously for 30 s. Add 0, 2, 4, 6, 8, and 10 µl of diluted 50 µM FITC Standard into a
series of wells in a 96-well white plate and adjust the final volume to 100 µl/well with Gelatinase Assay Buffer to generate 0, 100, 200,
300, 400, and 500 pmol/well of FITC Standard respectively. Mix well and measure the fluorescence at Ex/Em 490/520 nm in an end point
mode at 37 °C.
3. Gelatinase Substrate Mix: Prepare 50 µl of Gelatinase Substrate Mix per well as given below:
48 μl Gelatinase Assay Buffer
2 μl Reconstituted Gelatinase Substrate
Dissolve the Substrate Mix by vigorous vortexing. Add 50 µl of Substrate Mix solution into each Sample, and Positive Control well.
Note: Do not add Substrate Mix to the sample Background Control and Standard wells.
4. Measurement: Mix well and measure the fluorescence at Ex/Em 490/520 nm in kinetic mode at 37 °C for 1-2 h. Choose two time points
(t1 & t2) where the corresponding RFUs (RFU1 and RFU2) are in a linear range. Calculate ΔRFU and ∆t and obtain ΔRFU/∆t as RFU/min
for each Sample including background control. Subtract the value of RFU/min of background from each Sample to obtain net RFU/min
(B).
5. Calculations:
a. FITC Standard Curve: Obtain change in the RFU (ΔRFU) by subtracting fluorescence of the 0 Standard Controls from those containing
all standards. Plot the ΔRFU against pmol of FITC Standard. The plot should be linear; determine the slope A (ΔRFU/pmol) of the curve.
b. Samples: Using RFU/min of each Sample, calculate Sample Gelatinase activity using following equation.
𝐒𝐚𝐦𝐩𝐥𝐞 𝐆𝐞𝐥𝐚𝐭𝐢𝐧𝐚𝐬𝐞 𝐀𝐜𝐭𝐢𝐯𝐢𝐭𝐲 (𝑿,
𝐔
𝑩×𝟏𝟎𝟎𝟎
)=
×𝑫𝒊𝒍𝒖𝒕𝒊𝒐𝒏 𝑭𝒂𝒄𝒕𝒐𝒓
𝐦𝐥
𝑨×𝑪
𝐔
𝑿
𝐒𝐚𝐦𝐩𝐥𝐞 𝐆𝐞𝐥𝐚𝐭𝐢𝐧𝐚𝐬𝐞 𝐀𝐜𝐭𝐢𝐯𝐢𝐭𝐲 ( ) =
𝐦𝐠
𝑷
where, B = Sample Gelatinase Activity as calculated (RFU/min),
A = Slope of the FITC standard curve (ΔRFU/pmol),
C = µl of Sample used in the assay,
P = Protein concentration in the lysate (mg/ml).
1000 = Conversion Factor (1000 µl ≡ 1 ml)
Unit Definition: 1 U is the amount of Gelatinase required to cleave the Gelatinase Substrate and release 1 pmol of Fluorescein per min
under the conditions of the assay.
A
B
C
25000
20000
15000
10000
y = 24.445x + 1500.7
R² = 0.9891
5000
0
20 mCDU
Collagenase
15 mCDU
Collagenase
10 mCDU
Collagenase
8 mCDU
Collagenase
6 mCDU
Collagenase
4 mCDU
Collagenase
2 mCDU
Collagenase
No Enzyme
19000
17000
15000
13000
11000
9000
7000
5000
0
500
1000
FITC Standard (pmol)
0
20
40
Time (min)
U/mg
ΔRFU (Ex/Em = 490/520 nm)
30000
RFU (Ex/Em = 490/520 nm)
21000
100.00
90.00
80.00
70.00
60.00
50.00
40.00
30.00
20.00
10.00
0.00
60
Rat Heart
Rat Kidney
Hela Cells
U937 Cells
Figure: FITC Standard Curve (A), Gelatinase activity with different amounts of Enzyme Positive Control (B), and in rat heart, kidney lysates
along with Hela and U937 cell lysates (C) are shown in the figure (n = 3). The assays were performed according to the kit protocol.
VIII.
RELATED PRODUCTS:
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Collagenase Inhibitor Screening Kit (Fluorometric)
EZCell™ Cell Invasion Assay (Collagen IV), 96-well, 8 µm (K918)
EZCell™ Cell Invasion Assay (Collagen I), 96-well, 8 µm (K916)
MMP-9 Inhibitor Screening Kit (Fluorometric) (K844)
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Collagenase (Collagen Degradation/Zymography) Assay Kit (K490)
Collagenase Activity Colorimetric Assay Kit (K792)
Hydroxyproline Colorimetric Assay Kit (K555)
EZCell™ Cell Invasion Assay (Collagen IV), 24-well, 8 µm (K919)
EZCell™ Cell Invasion Assay Kit (Collagen I), 24-well, 8 µm (K917)
MMP-1 Inhibitor Screening Kit (Fluorometric)
Jurkat Cell Extract (Uninduced) (1106)
Jurkat Cell Extract (Induced) (1107)
FOR RESEARCH USE ONLY! Not to be used on humans.
155 S. Milpitas Blvd., Milpitas, CA 95035 USA | T: (408)493-1800 F: (408)493-1801 | www.biovision.com | [email protected]